PROJECT SUMMARY Clinical dementia of the Alzheimer's type (DAT) is the most common dementing disorder in the elderly. DAT is most tightly correlated with synaptic loss in vulnerable brain regions, which has led to the hypothesis that loss of synaptic terminals is a key event in early cognitive decline. To date, the mechanisms of synaptic loss in DAT remain unknown. Previous researchers have assumed that specific molecular entities such as tau or amyloid-beta (A?) are responsible for synaptic degeneration in DAT, but have not performed unbiased screens. Our primary hypothesis is there are as-yet- uncharacterized synaptotoxic substances in brain lysates from patients with DAT that are not present in brain lysates from high pathology, age matched, non-demented controls. We further hypothesize these substances cause synapses to deteriorate in the early phase of DAT which eventually leads to neuronal cell death and severe cognitive decline. To test our hypothesis, we have recently developed a novel, unbiased, high content screening approach using live primary hippocampal neurons from mice genetically manipulated to express a red fluorescent pre-synaptic tag and a green fluorescent post-synaptic tag. This new approach allows us to measure the synapse gain/loss of cultured live neurons serially over several days in a highly sensitive and fully quantitative fashion. We therefore propose to use our novel, unbiased screening approach to identify as-yet-uncharacterized synaptotoxic substances in brain lysates from patients with DAT and to further characterize the synaptotoxic activity of the newly-identified substances. Specific aims of the proposed studies are: 1) To determine whether there are synaptotoxic substances in aqueous brain lysates from patients with DAT using our novel live cell screening approach for synaptic toxicity, as well as how consistent the toxicity is from patient to patient; 2) To determine the specificity of the toxic entities from patients with DAT by assessing brain lysates from non-demented control subjects with A? and tau pathology, and normal control subjects; 3) To use our live neuron screening of fractionated aqueous brain lysates from patients with DAT for synaptic toxicity to guide purification of the most potent and specific toxic entities, and then identify the previously uncharacterized synaptotoxic substances in patients with DAT. If successful, we expect to identify a list of previously-uncharacterized synaptotoxic proteins, peptides or other substances specific to patients with DAT. Once the synaptotoxic activities of these substances have been characterized, we will target these novel synaptotoxic agents as new therapeutic targets and perform a drug screening for new candidate therapeutic molecules. The results of our proposed experiments may fundamentally advance knowledge of DAT as well as improve the effectiveness of DAT treatment by identifying targets for new candidate therapeutics.